This invention relates to Bacillus strains useful for the expression and secretion of desired polypeptides (as used herein, "polypeptide" means any useful chain of amino acids, including proteins).
Bacillus strains have been used as hosts to express heterologous polypeptides from genetically engineered vectors. The use of a Gram positive host such as Bacillus avoids some of the problems associated with expressing heterologous genes in Gram negative organisms such as E. coli. For example, Gram negative organisms produce endotoxins which may be difficult to separate from a desired product. Furthermore, Gram negative organisms such as E. coli are not easily adapted for the secretion of foreign products, and the recovery of products sequestered within the cells is time-consuming, tedious, and potentially problematic. In addition, Bacillus strains are non-pathogenic and are capable of secreting proteins by well-characterized mechanisms.
A general problem in using Bacillus host strains in expression systems is that they produce large amounts of proteases which can degrade heterologous polypeptides before they can be recovered from the culture media. The proteases which are responsible for the majority of this proteolytic activity are produced at the end of the exponential phase of growth, under conditions of nutrient deprivation, as the cells prepare for sporulation. The two major extracellular proteases an alkaline serine protease (subtilisin), the product of the apr gene, and a neutral metalloprotease, the product of the npr gene, are secreted into the medium, whereas the major intracellular serine protease, Isp-l, is produced within the cells. Other investigators have created genetically altered Bacillus strains that produce below-normal levels of one or more of these three proteases, but these strains still produce high enough levels of protease to cause the degradation of heterologous gene products prior to purification.
Stahl et al. (J. Bact., 1984, 158:411) disclose a Bacillus protease mutant in which the chromosomal subtilisin structural gene was replaced with an in vitro derived deletion mutation. Strains carrying this mutation produced only 10% of the wild-type extracellular serine protease activity. Yang et al. (J. Bact., 1984, 160:15) disclose a Bacillus protease mutant in which the chromosomal neutral protease gene was replaced with a gene having an in vitro derived deletion mutation. Fahnestock et al. (WO 86/01825) describe Bacillus strains lacking subtilisin activity which were constructed by replacing the native chromosomal gene sequence with a partially homologous DNA sequence having an inactivating segment inserted into it. Kawamura et al. (J. Bact., 1984, 160:442) disclose Bacillus strains carrying lesions in the npr and apr genes and expressing less than 4% of the wild-type level of extracellular protease activity. Koide et al. (J. Bact., 1986, 167:110) disclose the cloning and sequencing of the isp-l gene and the construction of an Isp-l negative mutant by chromosomal integration of an artificially deleted gene.
Genetically altered strains which are deleted for the extracellular protease genes (apr and npr) produce significantly lower levels of protease activity than do wild-type Bacillus strains. These bacteria, when grown on medium containing a protease substrate, exhibit little or no proteolytic activity, as measured by the lack of appearance of a zone of clearing (halo) around the colonies. Some heterologous polypeptides and proteins produced from these double mutants are, nevertheless, substantially degraded prior to purification, although they are more stable than when produced in a wild-type strain of Bacillus.